Type 1 diabetes (T1D) is an autoimmune disease that results in life-long insulin insufficiency, causes significant increases in morbidity and mortality, and is responsible for considerable social and economic costs. Thus, there is an urgent need for durable therapies that can prevent, arrest, or if possible reverse, the -cell loss that leads to T1D. Unfortunately, at present several factors are impeding progress towards a true cure. Foremost is the fact that the precise etiology of T1D in humans is still incompletely understood. This is compounded by a paucity of robust mechanistic biomarkers that can accurately reflect the current disease status in an individual patient. These are the critical gaps that our proposal seeks to address. Recent studies suggest a critical role for target tissues, including pancreatic cells, in initiating and/or propagating an autoimmune attack, and thus unwittingly contributing to their own demise. How could this happen? The central hypothesis is that cells, which are specialized to produce large quantities of insulin, are particularly sensitive to external factors that trigger cellular stress. Normal homeostatic responses to stress include both transcriptional and translational reprogramming. However, a likely consequence of this is the generation of polypeptides not normally present in cells that can be sources of cryptic neo-epitopes for autoreactive T cells. While several previous studies have examined the effects of cytokine exposure on human cells and detected changes in gene expression and splicing events, corresponding data on translational reprogramming is largely absent. Studies of protective immunity have shown that CD8+ T cells recognizing epitopes from non-canonical open reading frames (ORFs) are especially important in host responses to viruses, raising the possibility that they may also play a key role in autoimmunity. The primary goal of this proposal, therefore, is to define the unique spectrum of ?cryptic? translation products generated by stressed human cells (the ?Criptome?), and determine its potential as a source of pathogenic neo- epitopes. Three specific aims are proposed. First, we will determine the human cell ?Criptome? by ribosome profiling, using ribosome protected mRNA isolated from an immortalized human cell line, human pluripotent stem cell (hPSC) derived cells, and cadaveric human islets, at baseline and following exposure to cytokines. Second, we will validate our data using PUNCH-P, a complementary proteomics approach that uses mass spectrometry to analyze nascent polypeptides released from isolated polysomes by puromycin. Third, the newly identified cryptic ORFs will be interrogated for potential HLA-A2:01 epitopes, and the immunogenicity of the top candidates assessed using transgenic HLA-A2+ mice. If successful our rigorous approach will define a comprehensive cell translatome and CRiPtome, and identify novel biomarkers of human T1D, providing important new insight into both human cell biology, and the etiology of T1D.